Development of dendritic cells in the intestine

The intestinal tract is exposed to a large variety of antigens such as food proteins, commensal bacteria and pathogens and contains one of the largest arms of the immune system. The intestinal immune system has to discriminate between harmless and harmful antigens, inducing tolerance to harmless antigens and active immunity towards pathogens and other harmful materials. Dendritic cells (DC) in the mucosal lamina propria (LP) are central to this process, as they sample bacteria from the local environment and constitutively migrate to the draining mesenteric lymph nodes (MLN), where they present antigen to naïve T cells in order to direct an appropriate immune response. Despite their crucial role, understanding the function and phenotype of LP DC has been hampered by the fact that they share phenotypic markers with macrophages (mφ), which are the dominant population of mononuclear phagocyte (MP) in the LP. Recent work in our own and other laboratories has established gating strategies and phenotyping panels that allow precise discrimination between intestinal DC and mφ using the mφ specific markers CD64 and F4/80. In this way four bona fide DC subsets with distinct functions have been identified in adult LP based on their expression of CD11b and CD103 and a major aim of my project was to understand how these subsets might develop in the neonatal intestine. At the beginning of my PhD, the laboratory had used these new methods to show that signal regulatory protein α (SIRPα), an inhibitory receptor expressed by myeloid cells, was expressed by mφ and most DC in the intestine, except for those expressing CD103 alone. In addition, mice carrying a non-signalling mutation in SIRPα (SIRPα mt) had a selective reduction in CD103+CD11b+ DC, a subset which is unique to the intestinal LP. This was the basis for the initial experiments of my project, described in Chapter 3, where I investigated if the phenotype in SIRPα mt mice was intrinsic to haematopoietic cells or not. To explore this, I generated bone marrow (BM) chimeric mice by reconstituting irradiated WT mice with SIRPα mt BM, or SIRPα mt animals with WT BM. These experiments suggested that the defect in CD103+CD11b+ DC was not replicated in DC derived from BM of SIRPα origin. However as this seemed inconsistent with other data, I considered the possibility that 18 the phenotype may have been lost with age, as the BM chimeric mice were considerably older than those used in the original studies of SIRPα function. However a comparison of DC subsets in the intestine of WT and SIRPα mt mice as they aged provided no conclusive evidence to support this idea. As these experiments did show age-dependent effects on DC subsets, in Chapter 4, I went on to investigate how the DC populations appeared in the intestine and other tissues in the neonatal period. These experiments showed there were few CD103+CD11b+ DC present in the LP and migratory DC compartment of the MLN in the neonate and that as this population gradually increased in proportion with age, there was a reciprocal decrease in the relative proportion of CD103-CD11b+ DC. Interestingly, most of the changes in DC numbers in the intestine were found during the second or third week of life when the weaning process began. To validate my findings that there were few CD103+CD11b+ DC in the neonate and that this was not merely an absence of CD103 upregulation, I examined the expression of CD101 and Trem-1, markers that other work in the laboratory had suggested were specific to the CD103+CD11b+ DC lineage. My work showed that CD101 and Trem-1 were co- expressed by most CD103+CD11b+ DC in small intestine (SI) LP, as well as a small subset of CD103-CD11b+ DC in this tissue. Interestingly, Trem-1 was highly specific to the SI LP and migratory DC in the MLN, but absent from the colon and other tissues. CD101 expression was also only found on CD11b+ DC, but showed a less restricted pattern of distribution, being found in several tissues as well as the SI LP. The relative timing of their development suggested there might be a relationship between CD103+CD11b+ and CD103-CD11b+ DC and this was supported by microarray analysis. I hypothesised that the CD103-CD11b+ DC that co-expressed CD101 and Trem-1 may be the cells that developed into CD103+CD11b+ DC. To investigate this I analysed how CD101 and Trem-1 expression changed with age amongst the DC subsets in SI LP, colonic LP (CLP) and MLN. The proportion of CD101+Trem-1+ cells increased amongst CD103+CD11b+ DC in the SI LP and MLN with age, while amongst CD103+CD11b+ DC in the CLP this decreased. This was not the same in CD103-CD11b+ DC, where CD101 and Trem-1 expression was more varied with age in all tissues. CD101 and Trem-1 were not expressed to any great extent on CD103+CD11b- or CD103-CD11b- DC. The phenotypic development of the 19 intestinal DC subsets was paralleled by the gradual upregulation of CD103 expression, while the production of retinoic acid (RA), as assessed by the AldefluorTM assay, was low early in life and did not attain adult levels until after weaning. Thus DC in the neonatal intestine take some time to acquire the adult pattern of phenotypic subsets and are functionally immature compared with their adult counterparts. In Chapter 5, I used CD101 and Trem-1 to explore the ontogeny of intestinal DC subsets in CCR2-/- and SIRPα mt mice, both of which have selective defects in one particular group of DC. The selective defect seen amongst CD103+CD11b+ DC in adult SIRPα mt mice was more profound in mice at D7 and D14 of age, indicating that it may be intrinsic to this population and not highly dependent on environmental factors that change after birth. The expression of CD101 and Trem-1 by both CD103+CD11b+ and CD103-CD11b+ DC was reduced in SIRPα mt mice, again indicating that this entire lineage was affected by the lack of SIRPα signalling. However there was also a generalised defect in the numbers of all DC subsets in many tissues from early in life, suggesting there was compromised development, recruitment or survival of DC in the absence of SIRPα signalling. In contrast to the findings in SIRPα mt mice, more CD103+CD11b+ DC co-expressed CD101 and Trem-1 in CCR2-/- mice, while there were no differences in the expression of these molecules amongst CD103-CD11b+ DC. This may suggest that CCR2+ CD103-CD11b+ DC are not the cells that express CD101 and Trem-1 that are predicted to be the direct precursors of CD103+CD11b+ DC. I also examined the expression of DC growth factor receptors on DC subsets from mice of different ages, but no clear age or subset- related patterns of the expression of mRNA for Csf2ra, Irf4, Tgfbr1 and Rara could be observed. Next, I investigated whether Trem-1 played any role in DC development. Preliminary experiments in Trem-1-/- mice show no differences between any of the DC subsets, nor were there any selective effects on individual subsets when DC development from Trem-1-/- KO and WT BM was compared in competitive chimeras. However these experiments were difficult to interpret due to viability problems and because I found an unexpected defect in the ability of Trem-1-/- BM to generate all DC, irrespective of whether they expressed Trem-1 or not. 20 The final experiments I carried out were to examine the role of the microbiota in driving the differentiation of intestinal DC subsets, based on the hypothesis that this could be one of the environmental factors that might influence events in the developing intestine. To this end I performed experiments in both antibiotic treated and germ free adult mice, both of which showed no significant phenotypic differences amongst any of the DC subsets. However the study of germ free mice was compromised by recent contamination of the colony and may not be the conclusive answer. Together the data in this thesis have shown that the population of CD103+CD11b+ DC, which is unique to the intestine, is not present at birth. These cells gradually increase in frequency over time and as this occurs there is a reciprocal decrease in the frequency of CD103-CD11b+ DC. Along with other results, this leads to the idea that there may be a linear developmental pathway from CD103-CD11b+ DC to CD103+CD11b+ DC that is driven by non-microbial factors that are located preferentially in the small intestine. My project indicates that markers such as CD101 and Trem-1 may assist the dissection of this process and highlights the importance of the neonatal period for these events.

An investigation of the relationship between the components of tumour microenvironment, signal transduction pathways and outcome in breast cancer

Breast cancer, the most commonly diagnosed type of cancer in women, is a major cause of morbidity and mortality in the western world. Well-established risk factors of breast cancer are mostly related to women’s reproductive history, such as early menarche, late first pregnancy and late menopause. Survival rates have improved due to a combination of factors, including better health education, early detection with large-scale use of screening mammogram, improved surgical techniques, as well as widespread use of adjuvant therapy. At initial presentation, clinicopathological features of breast cancer such as age, nodal status, tumour size, tumour grade, and hormonal receptor status are considered to be the standard prognostic and predictive markers of patient survival, and are used to guide appropriate treatment strategies. Lymphovascular invasion (LBVI), including lymphatic (LVI) and blood (BVI) vessel invasion, has been reported to be prognostic and merit accurate evaluation, particularly in patients with node negative tumours who might benefit from adjuvant chemotherapy. There is a lack of standard assessment and agreement on distinguishing LVI from BVI despite the major challenges in the field. A systematic review of the literatures, examining methods of detection and the prognostic significance of LBVI, LVI and BVI, was carried out. The majority of studies used haematoxylin and eosin (H&E) and classical histochemistry to identify LVI and BVI. Only few recent studies used immunohistochemistry (IHC) staining of the endothelium lining lymphatic and blood vessels, and were able to show clear differences between LVI and BVI. The prognostic significance of LBVI and LVI was well-documented and strongly associated with aggressive features of breast tumours, while the prognostic value and the optimal detection method of BVI were unclear. Assessment and prognostic value of LBVI on H&E sections (LBVIH&E) was examined and compared to that of LVI and BVI detected using IHC with D2-40 for LVI (LVID2–40) and Factor VIII for BVI (BVIFVIII) in patients with breast cancer including node negative and triple negative patients (n=360). LBVIH&E, LVID2–40 and BVIFVIII were present in 102 (28%), 127 (35%) and 59 (16%) patients respectively. In node negative patients (206), LBVIH&E, LVID2–40 and BVIFVIII were present in 41 (20%), 53 (26%) and 21 (10%) respectively. In triple negative patients (102), LBVIH&E, LVID2–40 and BVIFVIII were present in 35 (29%), 36 (35%) and 14 (14%) respectively. LBVIH&E, LVID2–40 and BVIFVIII were all significantly associated with tumour recurrence in all cohorts. On multivariate survival analysis, only LVID2–40 and BVIFVIII were independent predictors of cancer specific survival (CSS) in the whole cohort (P=0.022 and P<0.001 respectively), node negative (P=0.008 and P=0.001 respectively) and triple negative patients (P=0.014 and P<0.001 respectively). Assessment of LVI and BVI by IHC, using D2-40 and Factor VIII, improves prediction of outcome in patients with node negative and triple negative breast cancer and was superior to the conventional detection method. Breast cancer is recognised as a complex molecular disease and histologically identical tumours may have highly variable outcomes, including different responses to therapy. Therefore, there is a compelling need for new prognostic and predictive markers helpful of selecting patients at risk and patients with aggressive diseases who might benefit from adjuvant and targeted therapy. It is increasingly recognised that the development and progression of human breast cancer is not only determined by genetically abnormal cells, but also dependent on complex interactions between malignant cells and the surrounding microenvironment. This has led to reconsider the features of tumour microenvironment as potential predictive and prognostic markers. Among these markers, tumour stroma percentage (TSP) and tumour budding, as well as local tumour inflammatory infiltrate have received recent attention. In particular, the local environment of cytokines, proteases, angiogenic and growth factors secreted by inflammatory cells and stromal fibroblasts has identified crucial roles in facilitating tumour growth, and metastasis of cancer cells through lymphatic and/or blood vessel invasion. This might help understand the underlying process promoting tumour invasion into these vessels. An increase in the proportion of tumour stroma and an increase in the dissociation of tumour cells have been associated with poorer survival in a number of solid tumours, including breast cancer. However, the interrelationship between these variables and other features of the tumour microenvironment in different subgroups of breast cancer are not clear. Also, whether their prognostic values are independent of other components of the tumour microenvironment have yet to be identified. Therefore, the relationship between TSP, clinicopathological characteristics and outcome in patients with invasive ductal breast cancer, in particular node negative and triple negative disease was examined in patients with invasive ductal breast cancer (n=361). The TSP was assessed on the haematoxylin and eosin-stained tissue sections. With a cut-off value of 50% TSP, patients with ≤50% stroma were classified as the low-TSP group and those with >50% stroma were classified as the high-TSP group. A total of 109 (30%) patients had high TSP. Patients with high TSP were old age (P=0.035), had involved lymph node (P=0.049), Her-2 positive tumours (P=0.029), low-grade peri-tumoural inflammatory infiltrate (P=0.034), low CD68+ macrophage infiltrate (P<0.001), low CD4+ (P=0.023) and low CD8+ T-lymphocytes infiltrate (P=0.017), tumour recurrence (P=0.015) and shorter CSS (P<0.001). In node negative patients (n=207), high TSP was associated with low CD68+ macrophage infiltrate (P=0.001), low CD4+ (P=0.040) and low CD8+ T-lymphocytes infiltrate (P=0.016) and shorter CSS (P=0.005). In triple negative patients (n=103), high TSP was associated with increased tumour size (P=0.017) high tumour grade (P=0.014), low CD8+ T-lymphocytes infiltrate (P=0.048) and shorter CSS (P=0.041). The 15-year cancer specific survival rate was 79% vs 21% in the low-TSP group vs high-TSP group. On multivariate survival analysis, a high TSP was associated with reduced CSS in the whole cohort (P=0.007), node negative patients (P=0.005) and those who received systemic adjuvant therapy (P=0.016), independent of other pathological characteristics including local host inflammatory responses. Therefore, a high TSP in invasive ductal breast cancer was associated with recurrence and poorer long-term survival. The inverse relation with the tumour inflammatory infiltrate highlights the importance of the amount of tumour stroma on immunological response in patients with invasive ductal breast cancer. Implementing this simple and reproducible parameter in routine pathological examination may help optimise risk stratification in patients with breast cancer. Similarly, the relationship between tumour budding, clinicopathological characteristics and outcome was examined in patients with invasive ductal breast cancer (n=474), using routine pathological sections. Tumour budding was associated with several adverse pathological characteristics, including positive lymph node (P=0.009), presence of LVI (P<0.001), and high TSP (P=0.001) and low-grade general peri-tumural inflammatory infiltrative (P=0.002). In node negative patients, a high tumour budding was associated with presence of LVI (P<0.001) and low-grade general peri-tumural inflammatory infiltrative (P=0.038). On multivariate survival analysis, tumour budding was associated with reduced CSS (P=0.001), independent of nodal status, tumour necrosis, CD8+ and CD138+ inflammatory cells infiltrate, LVI, BVI and TSP. Furthermore, tumour budding was independently associated with reduced CSS in node negative patients (P=0.004) and in those who have low TSP (P=0.003) and high-grade peri-tumoural inflammatory infiltrative (P=0.012). A high tumour budding was significantly associated with shorter CSS in luminal B and triple negative breast cancer subtypes (all P<0.001). Therefore, tumour budding was a significant predictor of poor survival in patients with invasive ductal breast cancer, independent of adverse pathological characteristics and components of tumour microenvironment. These results suggest that tumour budding may promote disease progression through a direct effect on local and distant invasion into lymph nodes and lymphatic vessels. Therefore, detection of tumour buds at the stroma invasive front might therefore represent a morphologic link between tumour progression, lymphatic invasion, spread of tumour cells to regional lymph nodes, and the establishment of metastatic dissemination. Given the potential importance of the tumour microenvironment, the characterisation of intracellular signalling pathways is important in the tumour microenvironment and is of considerable interest. One plausible signalling molecule that links tumour stroma, inflammatory cell infiltrate and tumour budding is the signal transducer and activator of transcription (STAT). The relationship between total and phosphorylated STAT1 (ph-STAT1), and total and ph-STAT3 tumour cell expression, components of tumour microenvironment and survival in patients with invasive ductal breast cancer was examined. IHC of total and ph-STAT1/STAT3 was performed on tissue microarray of 384 breast cancer specimens. Cellular STAT1 and cellular STAT3 expression at both cytoplasmic and nuclear locations were combined and identified as STAT1/STAT3 tumour cell expression. These results were then related to CSS and phenotypic features of the tumour and host. A high ph-STAT1 and a high ph-STAT3 tumour cell expression was associated with increased ER (P=0.001 and P<0.001 respectively) and PR (all P<0.05), reduced tumour grade (P=0.015 and P<0.001 respectively) and necrosis (all P=0.001). Ph-STAT1 was associated with increased general peri-tumoural inflammatory infiltrate (P=0.007) and ph-STAT3 was associated with lower CD4+ T-lymphocyte infiltrate (P=0.024). On multivariate survival analysis, including both ph-STAT1 and ph-STAT3 tumour cell expression, only high ph-STAT3 tumour cell expression was significantly associated with improved CSS (P=0.010) independent of other tumour and host-based factors. In patients with high necrosis grade, high ph-STAT3 tumour cell expression was independent predictor of improved CSS (P=0.021). Ph-STAT1 and ph-STAT3 were also significantly associated with improved cancer specific survival in luminal A and B subtypes. STAT1 and STAT3 tumour cell expression appeared to be an important determinant of favourable outcome in patients with invasive ductal breast cancer. The present results suggest that STATs may affect disease outcome through direct impact on tumour cells, and the surrounding microenvironment. The above observations of the present thesis point to the importance of the tumour microenvironment in promoting tumour budding, LVI and BVI. The observations from STATs work may suggest that an important driving mechanism for the above associations is the presence of tumour necrosis, probably secondary to hypoxia. Further work is needed to examine the interaction of other molecular pathways involved in the tumour microenvironment, such as HIF and NFkB in patients with invasive ductal breast cancer.